1. Field of the Invention
The invention relates to identification and characterization of a human interleukin-8 receptor which also binds gro. In another aspect, it relates to stable expression of functionally active IL-8 receptor in host cells.
2. Background Information
Stimulation of neutrophils with IL-8, NAP-2 or gro causes mobilization of intracellular calcium stores and elicits motile, secretory, and metabolic responses that are critical to the role of the neutrophil in host defenses. See B. Moser, I. Clark-Lewis, R. Zwahlen, M. Baggiolini, J. Exp. Med. 171, 1797 (1990); A. Walz, B. Dewald, V. von Tscharner, M. Baggiolini, ibid. 170, 1745 (1989); and M. Thelen et al., FASEB J. 2, 2702 (1988). IL-8 is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation and the respiratory burst, the means by which neutrophils attack pathogens in the body. Neutrophils express receptors for IL-8 that are coupled to guanine nucleotide binding proteins (G-proteins); binding of IL-8 to its receptor induces the mobilization of intracellular calcium stores. IL-8, also known as neutrophil activating protein-1 or NAP-1, is a potent chemoattractant for neutrophils that is produced by many cell types in response to inflammatory stimuli. See J. J. Oppenheim, Prog. Clin. Biol. Res. 349, 405 (1990). This IL-8 receptor has 77% amino acid identity with a second human neutrophil receptor isotype that also binds IL-8 (Genentech, FASEB, April 1991).
IL-8 is structurally and functionally related to several members of the macrophage inflammatory protein-2 (or MIP-2) family of cytokines. These include MIP-2, gro (or melanoma growth-stimulatory activity), and NAP-2. See S. D. Wolpe and A. Cerami, FASEB J. 3, 2565 (1989); B. Moser, I. Clark-Lewis, R. Zwahlen, M. Baggiolini, J. Exp. Med. 171, 1797 (1990); and A. Walz, B. Dewald, V. von Tscharner, M. Baggiolini, ibid. 170, 1745 (1989). High affinity binding sites for IL-8 have been found on transformed myeloid precursor cells such as HL60 and THP-1 as well as on neutrophils. See B. Moser, C. Schumacher, V. von Tscharner, I. Clark-Lewis, M. Baggiolini, J. Biol. Chem. 266, 10666 (1991); J. Besemer, A. Hujber, B. Kuhn, J. Biol. Chem. 264, 17409 (1989); P. M. Grob et al., ibid. 265, 8311 (1990); A. K. Samanta, J. J. Oppenheim, K. Matsushima, J. Exp. Med. 169, 1185 (1989); E. J. Leonard et al., J. Immunol. 144, 1323 (1989). NAP-2 and gro compete with IL-8 for binding to human neutrophils suggesting that they interact with the same receptors. See B. Moser, C. Schumacher, V. von Tscharner, I. Clark-Lewis, M. Baggiolini, J. Biol. Chem. 266, 10666 (1991).
Functional expression in the Xenopus oocyte has established the identity of cDNA clones encoding rabbit and human forms of another peptide chemoattractant receptor on neutrophils, the N-formyl peptide receptor. See K. M. Thomas, H. Y. Pyun, J. Navarro, J. Biol. Chem. 265, 20061 (1990); and F. Boulay, M. Tardif, L. Brouchon, P. Vignais, Biochem. Biophys. Res. Commun. 168, 1103 (1990). Yet the amino acid sequence of the rabbit form of the receptor (originally designated F3R) is only 28% identical with that of the human form (designated in this paper as FPR); this is far greater than the differences between species reported for all other G protein-coupled receptors. See T. I. Bonner, A. C. Young, M. R. Brann, N. J. Buckley, Neuron 1, 403 (1988); and S. Yokoyama, K. E. Eisenberg, A. F. Wright, Mol. Biol. Evol. 6, 342 (1989).
By cloning the complementary DNA sequence encoding the human interleukin 8 receptor (IL8R), the primary structure of this receptor can be established and its role in the inflammatory response can be further investigated. Such studies could potentially lead to the design of new anti-inflammatory agents.